title: Gradual disordering of the native state on a slow two-state folding protein monitored by single-molecule fluorescence spectroscopy and NMR.
abstract: Theory predicts that folding free energy landscapes are intrinsically malleable and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in a two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET) and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way in the sense that it converts between the native state and an unfolded ensemble that expands in response to chemical denaturants. However, as conditions become increasingly destabilizing, the native state also expands gradually, and a large fraction of its native intramolecular hydrogen bonds break up. This gradual disordering of the native state takes place in times shorter than the 100 μs resolution of our SM-FRET experiments. α-Spectrin SH3 thus showcases the extreme plasticity of folding landscapes, which extends to the native state of slow two-state proteins. Our results point to the idea that folding mechanisms under physiological conditions might be quite different from those obtained by linear extrapolation from denaturing conditions. Furthermore, they highlight a pressing need for re-evaluating the conventional procedures for analyzing and interpreting folding experiments, which may be based on too-simplistic assumptions.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas , Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Campos
authors.forename: Mourad
authors.initials: M
authors.lastname: Sadqi
authors.forename: Jianwei
authors.initials: J
authors.lastname: Liu
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
authors.forename: Douglas S
authors.initials: DS
authors.lastname: English
authors.forename: Victor
authors.initials: V
authors.lastname: Muñoz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D005456:Fluorescent Dyes / Q000737:chemistry; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D018909:src Homology Domains
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013049:Spectrin